Printed hyaluronic acid scaffolds

Abstract

The present invention relates a hydrogel comprising a porous structure of ordered structural elements, wherein said structural elements comprises crosslinked hyaluronic acid. The present invention also relates to the use of said hydrogels and methods for preparing said hydrogels.

1 . A hydrogel comprising a porous structure of ordered structural elements, wherein said structural elements comprises crosslinked hyaluronic acid. 
     
     
         2 . The hydrogel according to  claim 1 , wherein said hydrogel comprises multiple layers. 
     
     
         3 . The hydrogel according to any of  claims 1  and  2 , wherein said structural elements form a hierarchical structure. 
     
     
         4 . The hydrogel according to any of the preceding claims, wherein said porous structure comprises interconnected pores. 
     
     
         5 . The hydrogel according to  claim 4 , wherein said pores have a width of from 0.05 mm to 5 mm. 
     
     
         6 . The hydrogel according to  claim 5 , wherein said pores have a width of from 0.1 mm to 3 mm. 
     
     
         7 . The hydrogel according to any of the preceding claims, wherein said structural elements are fibres and/or particles. 
     
     
         8 . The hydrogel according to  claim 7 , wherein said fibres have a diameter in the range of from 50 μm to 1000 μm. 
     
     
         9 . The hydrogel according to  claim 8 , wherein said fibres have a diameter in the range of from 100 μm to 300 μm. 
     
     
         10 . The hydrogel according to  claim 9 , wherein said fibres have a diameter of about 200 μm. 
     
     
         11 . The hydrogel according to any of the preceding claims, comprising a plurality of parallel fibres. 
     
     
         12 . The hydrogel according to  claim 11 , comprising a plurality of parallel fibres within the same layer. 
     
     
         13 . The hydrogel according to any of the preceding claims, wherein said hydrogel comprises multiple layers and wherein fibres between neighbouring layers are not parallel. 
     
     
         14 . The hydrogel according to any of  claims 7 - 14 , wherein said fibres are straight. 
     
     
         15 . The hydrogel according to any of  claims 7 - 14 , wherein said fibres are coiled. 
     
     
         16 . The hydrogel according to any of  claims 7 - 14 , wherein said hydrogel comprises straight fibres and coiled fibres. 
     
     
         17 . The hydrogel according to  claim 17 , wherein at least one layer consists of or comprises alternating coiled and straight fibres. 
     
     
         18 . The hydrogel according to any of  claims 16 - 18 , wherein each of said coiled fibres forms a periodic curve. 
     
     
         19 . The hydrogel according to  claim 13 , wherein fibres between neighbouring layers form an angle of at least 20°. 
     
     
         20 . The hydrogel according to any of  claims 7 - 20 , wherein said particles have a diameter in the range of from 30 μm to 1 mm. 
     
     
         21 . The hydrogel according to any  claims 7 - 21 , wherein said particles are aligned in rows. 
     
     
         22 . The hydrogel according to any  claims 7 - 22 , wherein said hydrogel comprises multiple layers and wherein each layer comprises multiple rows of particles. 
     
     
         23 . The hydrogel according to  claim 23 , wherein said rows are parallel. 
     
     
         24 . The hydrogel according to any of the preceding claims, wherein said hyaluronic acid is crosslinked with DVS, BDDE and/or tyramine. 
     
     
         25 . The hydrogel according to any of the preceding claims, wherein said hydrogel is biocompatible. 
     
     
         26 . The hydrogel according to any of the preceding claims, further comprising at least one biologically active agent. 
     
     
         27 . The hydrogel according to any of the preceding claims, further comprising at least one additional component selected from the group consisting of calcium phosphates, synthetic water-soluble polymers, synthetic water insoluble polymers, plant derived carbohydrates, microbially derived carbohydrates, animal carbohydrates, extracellular matrix compounds, chitosan and derivatives thereof, fatty acids, soaps, micellar particles, amino acids, peptides, polypeptides, proteins, glycoproteins, proteoglycans, ribonucleic acids, glass, silicates, and insoluble ceramics. 
     
     
         28 . The hydrogel according to any of the preceding claims, further comprising cells. 
     
     
         29 . The hydrogel according to any of the preceding claims, wherein said hydrogel comprises a coating. 
     
     
         30 . The hydrogel according to  claim 30 , wherein, wherein said coating is fibronectin. 
     
     
         31 . The hydrogel according to any of the preceding claims, wherein said hydrogel is three-dimensional. 
     
     
         32 . A method of preparing a crosslinked HA hydrogel comprising the steps of
 a. mixing HA, DVS and solvent to obtain a mixture of said DVS, HA and solvent;   b. depositing said solution onto a cooled substrate to obtain a frozen construct;   c. contacting (or immersing) said frozen construct with an alkaline solution at a temperature below the freezing point of the construct to cross-link HA with DVS,   whereby a cross-linked hydrogel is obtained.   
     
     
         33 . The method according to  claim 32 , wherein said hydrogel is as defined in any of  claims 1 - 31 . 
     
     
         34 . The method according to any of  claims 32 - 33 , wherein said alkaline solution comprises salt at concentration of at least 100 mM. 
     
     
         35 . The method according to any of  claims 32 - 34 , wherein said alkaline solution comprises base at a concentration of at least 100 mM. 
     
     
         36 . The alkaline solution according to  claim 35 , wherein said base is selected from the group consisting of NaOH, LiOH, KOH, Mg(OH) 2 , Ca(OH) 2  Sr(OH) 2 , Ba(OH) 2 . 
     
     
         37 . The method according to any of  claims 32 - 36 , wherein said HA and said DVS are mixed before addition of solvent. 
     
     
         38 . The method according to any of  claims 32 - 37 , wherein said HA is in the form of a powder. 
     
     
         39 . The method according to any of  claims 32 - 38 , wherein the ratio of HA to DVS is in the range of from 10:1 to 2:1 by weight. 
     
     
         40 . The method according to  claim 39 , wherein the ratio of HA to DVS is in the range of from 5:1 to 3:1 by weight. 
     
     
         41 . The method according to any one of  claims 32 - 40 , wherein said solvent comprises a buffer. 
     
     
         42 . The method according to any one of the  claims 32 - 41 , wherein said solvent has a pH below 8. 
     
     
         43 . The method according to any one of  claims 32 - 42 , wherein said solvent comprises one or more salts. 
     
     
         44 . The method according to any one of  claims 32 - 43 , wherein said solvent comprises sodium and/or potassium ions. 
     
     
         45 . The method according to any one of  claims 32 - 44 , wherein said solvent comprises sodium and/or potassium ions in a concentration of at least 100 mM. 
     
     
         46 . The method according to any one of  claims 32 - 45 , wherein said substrate is cooled to a temperature below 0° C. 
     
     
         47 . The method according to  claim 46 , wherein said substrate is cooled to a temperature below −20° C. 
     
     
         48 . The method according to any one of  claims 32 - 47 , wherein said substrate is cooled using dry ice. 
     
     
         49 . The method according to any one of  claims 32 - 48 , wherein said alkaline solution comprises at least one additional component selected from the group consisting of calcium phosphates, synthetic watersoluble polymers, synthetic waterinsoluble polymers, plant derived carbohydrates, microbially derived carbohydrates, animal carbohydrates, extracellular matrix compounds, chitosan and derivatives thereof, fatty acids, soaps, micellar particles, amino acids, peptides, polypeptides, proteins, glycoproteins, proteoglycans, ribonucleic acids, glass, silicates, and insoluble ceramics. 
     
     
         50 . The method according to any one of  claims 32 - 49 , wherein said alkaline solution comprises gelatine. 
     
     
         51 . The method according to any one of  claims 32 - 50 , wherein said alkaline solution comprises hyaluronic acid. 
     
     
         52 . The method according to any one of  claims 32 - 51 , wherein said alkaline solution comprises silk fibroin. 
     
     
         53 . The method according to any one of  claims 32 - 52 , wherein said alkaline solution comprises at least one biologically active agent. 
     
     
         54 . The method according to any one of  claims 32 - 53 , wherein said alkaline solution comprises at least one drug carrier. 
     
     
         55 . The method according to any one of  claims 33 - 54 , wherein the construct is heated to the melting point before contacting with the alkaline solution. 
     
     
         56 . The method according to any one of  claims 33 - 55 , wherein the solution is deposited using at least two solutions thereby obtaining a hydrogel comprising compartments corresponding to the different solutions. 
     
     
         57 . The method according to any one of  claims 33 - 56 , wherein said at least two solutions comprise different concentrations of HA and DVS. 
     
     
         58 . The method according to any one of  claims 33 - 57 , wherein said alkaline solution has a pH>10. 
     
     
         59 . The method according to any one of  claims 33 - 58 , wherein said solution is deposited using a device comprising a motor-driven nozzle. 
     
     
         60 . The method according to any one of  claims 33 - 59 , wherein said solution is deposited using fused deposition modelling (FDM). 
     
     
         61 . The method according to any one of  claims 33 - 60 , wherein said solution is deposited in at least one layer. 
     
     
         62 . The method according to any one of  claims 33 - 61 , wherein said solution is deposited in a layer-by-layer process thereby obtaining a construct comprising one or more layers. 
     
     
         63 . The method according to any one of  claims 33 - 62 , further comprising a step of coating the printed construct or the hydrogel with a coating. 
     
     
         64 . The method according to any one of  claims 33 - 63  further comprising a step of washing the hydrogel to obtain a neutral pH of said hydrogel. 
     
     
         65 . The method according to  claim 64 , wherein said hydrogel is washed in water, in a buffer having a pH of about 7 and/or in a solution comprising water and alcohol. 
     
     
         66 . The method according to any one of  claims 33 - 65 , wherein said hydrogel is washed at least two times. 
     
     
         67 . The method according to  claim 66 , comprising a step of washing the hydrogel in water and/or in a buffer having a pH of about 7 and further comprising a step of washing the hydrogel in a solution comprising water and alcohol. 
     
     
         68 . A method preparing a crosslinked HA hydrogel comprising the steps of
 a. mixing HA, BDDE and a first solvent to obtain a mixture of said HA, BDDE and first solvent;   b. depositing said solution onto a cooled substrate to obtain a frozen construct;   c. lyophilizing the frozen construct to obtain a lyophilized construct;   d. immersing said lyophilized construct in a second solvent   
       whereby a cross-linked hydrogel is obtained. 
     
     
         69 . The method according to  claim 68 , wherein said HA and BDDE are mixed before addition of the first solvent. 
     
     
         70 . The method according to any of  claims 68 - 69 , wherein said HA is in the form of a powder. 
     
     
         71 . The method according to any of  claims 68 - 70 , wherein said frozen construct is embedded in a second solution comprising HA before lyophilizing said frozen construct. 
     
     
         72 . The method according to any one of  claims 68 - 71 , wherein the ratio of HA to BDDE is in the range of from 10:1 to 2:1 by weight. 
     
     
         73 . The method according to  claim 72 , wherein the ratio of HA to BDDE is in the range of from 5:1 to 3:1 by weight. 
     
     
         74 . The method according to any one of  claims 68 - 73 , wherein said first solvent is water. 
     
     
         75 . The method according to any one of  claims 68 - 73 , wherein said first solvent is a buffer. 
     
     
         76 . The method according to any one of  claims 68 - 75 , wherein said first solvent comprises one or more salts. 
     
     
         77 . The method according to  claim 76 , wherein said first solvent comprises sodium and/or potassium ions. 
     
     
         78 . The method according to any one of  claims 76 - 77 , wherein said first solvent comprises salt in a concentration of at least 100 mM. 
     
     
         79 . The method according to any one of  claims 68 - 78 , wherein said second solvent is water. 
     
     
         80 . The method according to any one of  claims 68 - 78 , wherein said second solvent is a buffer. 
     
     
         81 . The method according to any one of  claims 69 - 80 , wherein said second solvent comprises one or more salts. 
     
     
         82 . The method according to  claim 81 , wherein said second solvent comprises sodium and/or potassium ions. 
     
     
         83 . The method according to any one of  claims 81 - 82 , wherein said second solvent comprises salt in a concentration of at least 100 mM. 
     
     
         84 . The method according to any one of  claims 68 - 83 , wherein said second solvent comprises at least one additional component selected from the group consisting of calcium phosphates, synthetic water-soluble polymers, synthetic water insoluble polymers, plant derived carbohydrates, microbial derived carbohydrates, animal carbohydrates, extracellular matrix compounds, chitosan and derivatives thereof, fatty acids, soaps, micellar particles, amino acids, peptides, polypeptides, proteins, glycoproteins, proteoglycans, ribonucleic acids, glass, silicates, and insoluble ceramics. 
     
     
         85 . The method according to any one of  claims 68 - 84 , wherein said second solvent comprises gelatine. 
     
     
         86 . The method according to any one of  claims 68 - 85 , wherein said second solvent comprises hyaluronic acid. 
     
     
         87 . The method according to any one of  claims 68 - 86 , wherein said second solvent comprises silk fibroin. 
     
     
         88 . The method according to any one of  claims 68 - 87 , wherein said second solvent comprises at least one drug carrier. 
     
     
         89 . The method according to any one of  claims 68 - 88 , wherein said second solvent comprises at least one biologically active agent. 
     
     
         90 . The method according to any one of  claims 68 - 89 , wherein the solution is deposited using at least two solutions thereby obtaining a hydrogel comprising compartments corresponding to the different solutions. 
     
     
         91 . The method according to any one of  claims 68 - 90 , wherein said at least two solutions comprise different concentrations of HA and BDDE. 
     
     
         92 . The method according to any one of  claims 68 - 91 , wherein said solution is deposited using a device comprising a motor-driven nozzle. 
     
     
         93 . The method according to any one of  claims 68 - 92 , wherein said solution is deposited using fused deposition modelling (FDM). 
     
     
         94 . The method according to any one of  claims 68 - 93 , wherein said solution is deposited in at least one layer. 
     
     
         95 . The method according to any one of  claims 68 - 94 , wherein said solution is deposited in a layer-by-layer process thereby obtaining a construct comprising one or more layers. 
     
     
         96 . The method according to any one of  claims 68 - 95 , further comprising a step of coating the printed construct or the hydrogel with a coating. 
     
     
         97 . The method according to any one of  claims 32 - 96 , wherein said construct is three-dimensional. 
     
     
         98 . Use of a hydrogel prepared by the method according to any one of  claims 32 - 96  or the hydrogel according to any one of  claims 1 - 31 , for making a 3D cell culture matrix. 
     
     
         99 . The use according to  claim 98 , wherein said 3D cell culture matrix is used for tissue repair and/or cell therapy. 
     
     
         100 . Use of a hydrogel prepared by the method according to any one of  claims 32 - 96  or the hydrogel according to any one of  claims 1 - 31 , for cosmetic surgery, wound dressing, drug development, stem cell research and/or cancer research 
     
     
         101 . A cross-linked HA hydrogel obtainable by the method according to any one of  claims 32 - 96 . 
     
     
         102 . The cross-linked HA hydrogel according to  claim 101 , wherein said hydrogel has a loss modulus of at least 10 Pa. 
     
     
         103 . The cross-linked HA hydrogel according to any one of  claims 101 - 102 , wherein said hydrogel has storage modulus greater than the loss modulus. 
     
     
         104 . A 3D cell culture matrix comprising the hydrogel according to any one of  claims 1 - 31  or  claims 101 - 103 . 
     
     
         105 . A wound dressing comprising the 3D cell culture matrix according to  claim 104 .